To advance our long-term goal of understanding the regulation of immunoglobulin isotype switching in human B lymphocytes, we have been studying control regions, including a known enhancer complex, that lie downstream of the human and murine immunoglobulin C-alpha genes and that may play a role in isotype switching induced by cytokines and CD40 engagement. We use several technologies to determine how these downstream regions integrate developmental maturation and signals from the B cell milieu to regulate Ig expression. We have stably transfected several cell lines with GFP constructs linked to various segments of these control regions region to test for position-independent copy-number-dependent expression indicative of LCR (Locus Control Region) function. We have mapped Dnase I hypersensitive sites in the 60 kb downstream of the immunoglobulin C-alpha gene, identifying several regions of modified chromatin context. We have used chromatin immunoprecipitation (ChIP) assays to identify several regions downstream of the immunoglobulin heavy chain locus that are associated in vivo with transcription factors or modified histone proteins. We have also identified candidate insulator DNA fragments by their ability to block in vivo the interaction between enhancer and promoter of stably tranfected reporter constructs. Taken together, these findings suggest the existence of a gene insulator downstream of the immunoglobulin heavy chain gene locus that might function to prevent crosstalk between the enhancers controlling the IgH locus and the regulatory sequences associated with the next closest non-immunoglobulin gene. These studies should help clarify the regulation of immunoglobulin gene expression. In a related project we have been exploring the molecular mechanism of immunoglobulin isotype switching. Previous studies have detected blunt double-stranded cuts in immunoglobulin switch regions undergoing switch recombinations, though several investigators have hypothesized that staggered cuts, rather than blunt, initiate the switch recombination. We have studied CH12F3, a murine B cell line capable of switching in vitro from IgM to IgA when stimulated with IL4, TGF-beta and CD40 Ligand. Using a ligation-mediated PCR procedure we have detected single-strand non-blunt DNA breaks that occur at specific reproducible positions in the switch region of the alpha locus, and that are dependent on incubating the cells under conditions that cause switching to this locus. These breaks likely represent intermediates in the switch recombination, and should help clarify the mechanism of this important process in the regulation of specific isotype expression.